Sugar-regulatory sequences in alpha-amylase genes

ABSTRACT

A chimeric plant promoter which is responsive to sugar depletion or deprivation is disclosed. The promoter includes a promoter element and, carried in the element, a heterologous sequence from the rice Amy3D that is responsive to sugar depletion. Also disclosed is a plant transfecting vector containing the sequence, and plant cell transfected with the vector.

This application is a continuation of U.S. patent application Ser. No.09/450,515, filed Nov. 29, 1999, now U.S. Pat. No. 6,680,425, issuedJan. 20, 2004 [now pending]; which is a continuation of application Ser.No. 09/046,858, filed Mar. 24, 1998, now U.S. Pat. No. 6,048,973, issuedApr. 11, 2000; which claims the priority of U.S. Provisional ofApplication No. 60/042,376; filed Mar. 24, 1997, now abandoned; whichare incorporated herein by reference.

This invention was made with Government support under Grant No. IBN94-08369, awarded by the National Science Foundation. The Government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to novel plant chimeric promoters that areresponsive to sugar depletion conditions in a plant cell.

BACKGROUND OF THE INVENTION

The Amy3D and Amy3E genes are two sugar-regulated genes in the riceα-amylase family (Huang, N. et al., Nucleic Acids Res., 18(23):7077(1990)). Amy3D is expressed in seedlings in response to sugar depletionand is not expressed when sugar, such as glucose, concentrations areelevated. Further, Amy3D is upregulated strongly in cell culture underconditions of sugar depletion or sugar deprivation. Unlike many otherα-amylase genes, Amy3D is not induced in response to gibberellic acid.

Metabolic regulation of Amy3D expression provides a signal mechanism tohelp control sugar production in the source tissues of germinatingcereal seedling. Thus, the rate of starch breakdown is modulated inresponse to the rate at which the embryo axis can utilize sugar for itsgrowth.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a chimeric plant promoter forupregulating expression of a coding sequence operatively linked to thepromoter, under conditions of sugar depletion or deprivation in a plantcell. The promoter includes a promoter element effective to express thecoding sequence, under selected conditions and, carried in the promoterelement, one or more of the following heterologous sequences which areresponsive to (upregulated by) sugar depletion in plant cells: SEQ IDNO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, and combinations of thesesequences. An exemplary combination is produced from sequences from SEQID NOS: 2 and 3, identified as SEQ ID NO:5.

In one embodiment the heterologous sequences is duplicated. In anotherembodiment, the promoter element is a monocot amylase-gene promoter, andthe heterologous sequence is inserted in the promoter element at aposition homologous to the position of the heterologous sequence in therice Amy3D gene. Exemplary promoter elements are from the RAmy1A,RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, pM/C, gKAmy141, gKAmy155,Amy32b, and HV18 genes.

Also forming part of the invention is a method of enhancing theinducibility by sugar depletion or deprivation of a plant or plant-viruspromoter in plant cells. The method includes introducing into a plantpromoter or plant-virus promoter, one or more of the above heterologoussequences, to achieve at least a 2-fold greater sugar-depletioninduction level over that of the unmodified promoter.

The promoter may be, for example, an α-amylase promoter from a monocotα-amylase gene, such as the RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B,RAmy3C, pM/C, gKAmy141, gKAmy155, Amy32b, or HV18 genes. The promotermay be duplicated in the chimeric promoter. The plant may be a monocotplant.

In another aspect, the invention includes a vector for use intransforming a plant. The vector includes a chimeric gene having,operatively linked in sequence in a 5′ to 3′ direction, (i) the abovechimeric plant promoter, (ii) a gene encoding a protein to be expressed,and (iii) a 3′ untranslated terminator region. Also disclosed are plantcells transfected with the vector.

These and other objects and features of the invention will be more fullyunderstood when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows metabolic signals and gibberellic acid (GA) signals in theregulation of cereal seed development;

FIG. 2A shows the p3DG expression plasmid for testing expression ofvarious deletions and site-specific mutations in regions of the RAmy3Dpromoter operably linked to the structural gene forβ-glucuronidase(GUS);

FIG. 2B shows that glucose (glc) treatment reduces expression, whilemannitol (man) treatment (sugar depletion) does not affect expressionfrom the RAmy3D promoter, as indicated by GUS activity in rice celllines transformed with the RAmy3D/GUS expression plasmid of FIG. 2A;

FIG. 3A shows a series of 5′ deletion constructs of the RAmy3D promoterincorporated into the GUS expression plasmid;

FIG. 3B shows levels of GUS expression observed in mannitol-treatedtransformed cell lines containing each of the series of RAmy3Ddeletions;

FIG. 4A shows the RAmy3D promoter in the region between nucleotides −207and −98 and site-directed mutations of the promoter where nucleotidesmatching the native promoter are indicated with (−), the native promoterin the −207 to −98 region having the sequence SEQ ID NO:6 and themutated promoter regions SDM#1 through SDM#10 having the sequences SEQID NO:7 through SEQ ID NO:16, respectively;

FIG. 4B shows levels of transient GUS expression observed inmannitol-treated (dark bars) and glucose-treated (light bars) riceprotoplasts transformed with expression plasmids containing themutations shown in FIG. 4A;

FIG. 5A shows the RAmy3D promoter in the region between nucleotides −166and −147 identified as a sugar-responsive region of the promoter, andsite-directed mutations within the region, the native promoter in thatregion having the sequence SEQ ID NO:5 and the mutated regions havingthe sequence SEQ ID NO:17 (SDM#5-1), SEQ ID NO:18 (SDM#5-2), SEQ IDNO:19 (SDM#6-1), SEQ ID NO:20 (SDM#6-2) and SEQ ID NO:21 (SDM#6-3);

FIG. 5B shows levels of transient GUS expression observed inmannitol-treated (dark bars) and glucose-treated (light bars) riceprotoplasts transformed with an expression plasmid containing each ofthe mutations shown in FIG. 5A.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms below have the following meaning, unless indicated otherwisein the specification.

“Cell culture” refers to cells and cell clusters, typically calluscells, growing on or suspended in a suitable growth medium.

“Germination” refers to the breaking of dormancy in a seed and theresumption of metabolic activity in the seed, including the productionof enzymes effective to break down starches in the seed endosperm.

“Inducible” means a promoter that is upregulated by the presence orabsence of a small molecule. It includes both indirect and directinducement.

“Inducible during germination” refers to promoters which aresubstantially silent but not totally silent prior to germination but areturned on substantially (greater than 25%) during germination anddevelopment in the seed.

“Inducible by sugar depletion” or “inducible by sugar deprivation”refers to promoters which are substantially silent or operate at arelatively low level in the presence of sugars such as those definedbelow, but are upregulated substantially (greater than 25% increase inexpression) in the absence of or in low levels of sugars, in either thegerminating seed or in cell culture. In cell culture, the sugardepletion induction response level may be measured by comparing thedifference in promoter activity when cells are cultured in the presenceof 3% glucose vs. 0% glucose (or alternatively, 3% mannitol). Examplesof promoters that are inducible by sugar depletion include promotersfrom the RAmy3D and RAmy3E rice α-amylase genes.

“Sugars”, as used herein, refer to sugars that are capable of repressingor inhibiting the transcriptional activity of the RAmy3D promoter (andother sugar-repressible promoters), and include any monosaccharide ordisaccharide that is capable of producing ATP by the glycolytic pathwayvia initial phosphorylation of the sugar. Examples include, but are notlimited to, glucose, sucrose, maltose, fructose, or otherphosphorylatable sugar. Sugar alcohols like mannitol, sorbitol, andglycerol, which are not capable of producing ATP via initialphosphorylation and entry into the glycolytic pathway, do not repressthe transcriptional activity of the RAmy3D promoter, and are thus notconsidered “sugars” in the context of this invention.

“Small molecules”, in the context of promoter induction, are typicallysmall organic or bioorganic molecules less than about 1 kilodalton.Examples of such small molecules include sugars, sugar-derivatives(including phosphate derivatives), and plant hormones (such as,gibberellic or absissic acid).

“Specifically regulatable” refers to the ability of a small molecule topreferentially affect transcription from one promoter or group ofpromoters (e.g., the α-amylase gene family), as opposed to non-specificeffects, such as, the enhancement or reduction of global transcriptionwithin the cell by, for example, increases or decreases in celldivision, cell size, or metabolic rate.

A “transcription regulatory region” or “promoter” refers to nucleic acidsequences that influence and/or promote initiation of transcription.Promoters are typically considered to include regulatory regions, suchas enhancer or inducer elements.

“Operably linked” refers to components of a chimeric gene or anexpression cassette that function as a unit to express a heterologousprotein. For example, a promoter operably linked to a heterologous DNA,which encodes a protein, promotes the production of functional mRNAcorresponding to the heterologous DNA.

A “promoter element” refers to a plant or plant-virus promoter which iseffective to express a coding sequence operatively linked the promoterunder selected conditions in plant cells. A promoter element may be anative plant promoter, e.g., an gene promoter from monocots, or dicots,a plant-virus promoter, e.g., the 35S CMV promoter, or a hybrid plant orplant-virus promoter, that is, a promoter constructed of elements fromvarious promoters. In some contexts, “promoter element” and “promoter”are used interchangeably.

A “sugar-regulatory sequence” or “sugar-depletion responsive sequence”is a sequence of nucleotides taken from the rice Amy3D promoter that hasbeen discovered, in accordance with the invention, to confer ability ofthe promoter to upregulate promoter activity, e.g., expression of a geneoperatively linked to the promoter, under conditions of sugar depletionor deprivation. The sugar-regulatory sequences include those defined bySEQ ID NOS: 1, 2, 3, 4 and combinations thereof.

“Sugar depletion” refers to utilization and depletion of existing sugarin plant cells or in the plant cell environment, by plant cellmetabolism.

“Sugar deprivation” means that plant cells are grown in the absence ofor at low concentrations of sugar.

A “heterologous sequence”, in the context of a sugar-regulatorysequence, means a sugar-regulatory sequence that is placed in a promoterelement (i) that normally does not contain such sequence, (ii) induplicate copies in a promoter element that may or may not contain sucha sequence in native form, and/or (iii) at promoter regions normally notcontaining the heterologous sequence.

A “homologous position”, in the context of a promoter region, refers toa position in a plant promoter gene, preferably a monocot α-amylasegene, that corresponds to the position in a rice Amy3D gene, when thetwo promoters are aligned.

A plant promoter is aligned with the rice Amy3D promoter sequence whenthe two sequences are optimally aligned, meaning the alignment givingthe highest % identity score between the sequences. Such alignment canbe preformed using a variety of commercially available sequence analysisprograms, such as the “GENEWORKS” program, or the local alignmentprogram LALIGN with a ktup of 1, default parameters and the default PAM.A preferred alignment is the one performed by the CLUSTAL-W program fromMacVector (TM), operated with an open gap penalty of 10.0, an extendedgap penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to beinserted into a first sequence to optimally align it with a secondsequence, the percent identity is calculated using only the residuesthat are paired with a corresponding amino acid residue (i.e., thecalculation does not consider residues in the second sequences that arein the “gap” of the first sequence).

“Heterologous DNA” or “foreign DNA” refers to DNA which has beenintroduced into plant cells from another source, or which is from aplant source, including the same plant source, but which is under thecontrol of a promoter or terminator that does not normally regulateexpression of the heterologous DNA.

“Heterologousprotein” is a protein, including a polypeptide, encoded bya heterologous DNA.

A “product” encoded by a DNA molecule includes, for example, RNAmolecules and polypeptides.

“Removal” in the context of a metabolite includes both physical removalas by washing and the depletion of the metabolite through the absorptionand metabolizing of the metabolite by the cells.

“Substantially isolated” is used in several contexts and typicallyrefers to the at least partial purification of a protein or polypeptideaway from unrelated or contaminating components. Methods and proceduresfor the isolation or purification of proteins or polypeptides are knownin the art.

“Stably transformed” as used herein refers to a cereal cell or plantthat has foreign nucleic acid stably integrated into its genome which istransmitted through multiple generations.

A DNA sequence is “derived from” a gene, such as a rice or barleyα-amylase gene, if it corresponds in sequence to a segment or region ofthat gene. Segments of genes which may be derived from a gene includethe promoter region, the 5′ untranslated region, and the 3′ untranslatedregion of the gene.

II. Chimeric, Sugar-Depletion Inducible Promoters

The metabolic regulation signals and plant hormone signals that controlcereal seedling development in rice are diagrammed in FIG. 1. As seen,synthesis of the rice α-amylase protein Amy3E is stimulated by sugardepletion in the seed aleurone cells, and Amy1A α-amylase production isstimulated by the plant hormone gibberellic acid (GA). In the scutellumlayer, sugar depletion induces synthesis of the Amy3D α-amylase. Theα-amylases are secreted into the endosperm of the seed, resulting inconversion of starch to sugar, which in turn represses Amy3D and Amy3Esynthesis. The sucrose produced is made available to the embryo forembryo growth.

A similar response is observed in rice cell culture. Amy3D is expressedwhen the cells are deprived of metabolizable sugars (i.e., grown in theabsence of metabolizable sugars such as glucose, sucrose, maltose,fructose, galactose) or in response to sugar depletion in the growthmedium. The converse is also true. When sugar concentrations areelevated in rice cell culture, Amy3D expression is reduced (Rodriguez,R., WO 95/14099; Yu S -M, et al. (1991) J. Biol. Chem. 266:21131-21137).

A. Sugar-Depletion Responsive Sequences

By deletion and site-directed mutation analyses described below and inthe Examples, Amy3D promoter sequence elements responsive to sugardepletion have been defined.

The ability of sugar depletion to upregulate Amy3D expression isdemonstrated in FIGS. 2A and 2B. FIG. 2A shows a plant transformationvector, designated p3DG, designed for transformation of monocots,containing the GUS gene under the control of the RAmy3D promoter(Example 1B). The plasmid p3DG contains 876 bp of RAmy3D 5′ flankingregion plus 66 bp of the 5′ untranslated leader sequence linked to theGUS coding region (FIG. 2A). The GUS gene encodes the enzymebeta-glucuronidase that produces a blue chromophore in tissuesexpressing the gene.

Rice plant cells were transformed with the vector according to standardmethods, such as detailed in PCT application WO 95/14099, which isincorporated herein by reference, and is described in Example 1C.Successful transformants were then grown in suspension culture undersugar-depletion vs. glucose treatment conditions, both in the presenceand absence of GA. Mannitol is a non-metabolizable sugar alcohol whichdoes not interact with the RAmy3D promoter, and is used insugar-depletion experiments to provide an osmotic strength equivalent tothat of the glucose treatment experiments. FIG. 2B shows the levels ofGUS expression observed, shown as GUS activity (pmole/min/mg tissue).GUS expression in transformed cells cultured under sugar-depletionconditions (i.e. in the presence of mannitol), either +/−GA, was atleast 10-15 times higher than in cells cultured in glucose-containingmedia.

The 5′ DNA region between nucleotides −876 to +66 (where +1 is the siteof initiation of transcription) of the Amy3D promoter contains all ofthe regulatory sequences necessary for metabolic regulation of Amy3Dtranscription. In a series of deletions proceeding from the 5′ end ofthe Amy3D promoter, diagrammed in FIG. 3A, the first sharp decline insugar depletion-induced GUS expression in the transformed cell linesoccurred upon deletion of the region between nucleotides −206 and −98(FIG. 3B, Example 1E.1), indicating that this region contains sequencescritical for high-level expression of the Amy3D promoter under sugardepletion conditions.

This region was examined in greater detail using full-length Amy3Dpromoters containing the short mutated segments shown in FIG. 4A. Levelsof GUS expression in a transient expression system (Example 1D) as afunction of mutated promoter segment were measured, with resultspresented in FIG. 4B and Example 1E.2.

The promoter sequences critical for sugar-depletion response in theAmy3D promoter were localized to the region having the sequenceidentified herein as SEQ ID NO:1, which extends between nucleotides −166and −125 of the Amy3D gene. In particular, three critical sequences havebeen identified: a) between nucleotides −166 and −157 and identified asSEQ ID NO:2; b) between nucleotides −155 and −147 and identified as SEQID NO:3; and c) between nucleotides −132 and −125 and identified hereinas SEQ ID NO:4.

The Amy3D sequence(s) critical for upregulation under sugar-depletionconditions (SEQ ID NOS:1-4 and combinations thereof) may be used tointroduce sugar-depletion regulation into plant promoters not naturallyregulated by sugar concentration, and to enhance or modulate thesugar-depletion response, e.g., by introducing multiple copies of thesequence into a plant promoter, for example a plant α-amylase genepromoter.

Combinations of these elements, such as the sequence between nucleotides−166 and −147 and identified herein as SEQ ID NO:5, are alsocontemplated. Also contemplated are sugar-depletion response sequencesthat are identical to the sequences disclosed herein, as well assequence elements which contain a small number of point mutations,deletions, or insertions, which do not compromise the induction ofexpression under sugar-depletion conditions.

B. Chimeric Promoters

The invention includes, in one aspect, a chimeric promoter containingone or more of the sugar-depletion responsive sequences identifiedabove. The promoter is designed for upregulating expression of a codingsequence operatively linked to the promoter, under conditions of sugardepletion or deprivation in a plant cell. The promoter includes (i) apromoter element effective to express the coding sequence in plantcells, under selected conditions, and (ii) carried in said promoterelement, one or more or the above heterologous sugar-depletionregulatory sequences identified as SEQ ID NO:1, SEQ ID NO: 2, SEQ IDNO:3, SEQ ID NO:4, and combinations thereof.

Various embodiments of the chimeric promoter are described below, withreference to methods of preparing such promoters. These methods are alsodescribed in the context of another aspect of the invention: a method ofenhancing the sugar-depletion response of a plant or plant-viruspromoter, by introducing one or more of the sugar-depletion regulatorysequences into the promoter element. The method is effective to increasethe sugar-depletion induction level of the chimeric promoter at least2-fold over the sugar-depletion induction level of the unmodified genepromoter.

Suitable promoters include those that transcribe the cereal α-amylasegenes and are induced by small molecules, including phytohormones suchas gibberellic acid or absissic acid, or by sugar depletion.Representative promoters include the promoters from the rice α-amylaseRAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, RAmy3D, and RAmy3Egenes, and from the pM/C, gKAmy141, gKAmy155, Amy32b, and HV18 barleyα-amylase genes. Such promoters, and their method of selection aredetailed in above-cited PCT application WO 95/14099.

Other suitable plant promoters include monocot and dicot plantpromoters, including, but not limited to, the actin, ubiquitin, Adh, Em,Lea, glutellin, hordein, zein, and Rubisco promoters. Alsocontemplatedare plant-viruspromoters, such as the 35S CMV promoter.

The sugar-depletion responsive sequence of the present invention may besubstituted for regulatory sequences in the promoters of other plantgenes, such as plant α-amylase genes. For example, a sugar-depletionresponsive sequence of the present invention may be substituted for theGA responsive element (GARE), or the abscisic acid responsive element(ABRE), in promoters which are induced by GA or abscisic acid,respectively, converting these promoters from phytohormone-regulatedpromoters to metabolically-regulated(i.e., sugar-depletion regulated)promoters.

Alternatively, or in addition, sugar-depletion responsive elements mayalso be substituted into structurally similar regions of other plantpromoters, including but not limited to monocot α-amylase promoters. Byaligning the sequences of the sugar-depletion responsive sequences withthe sequence of the target promoter, regions in the target promoterwhich are homologous to the sugar depletion-responsive element may beidentified. Such alignments may be performed using any of a number ofreadily-available sequence alignment programs, including the CLUSTAL Wprogram (MacVector(TM); Oxford Molecular, Oxford, UK). Promoterscontemplated include, but are not limited to, the GA-inducible RAmy1A,Amy32b, and HV18 promoters.

Alternatively, one or more sugar-depletion responsive elements of thepresent invention may be introduced into non-essential region(s) of aplant promoter. The non-essential regions of these promoters may beidentified using the experimental methods detailed herein, e.g.,locating sequence regions of the promoter which, when mutated, showpromoter activity substantially similar to that of the wildtypepromoter.

In another embodiment, the sugar-depletion responsive sequences of thepresent invention may be introduced into promotors which in their nativeform are upregulated by sugar depletion, to further enhance thesugar-depletion induction response of these promoters. Such promotersinclude the rice α-amylase RAmy3D and RAmy3E promoters. For example, itis contemplated that the presence of multiple copies of thesugar-depletion responsive sequences will enhance the sugar depletioninduction response of the RAmy3D or RAmy3E promoters.

III. Chimeric Gene

In another aspect, the invention includes a vector for use intransforming a monocotyledenous plant. The vector comprises a chimericgene having, operatively linked in sequence in a 5′ to 3′ direction, (i)a chimeric promoter as described above, (ii) operatively linked to thechimeric promoter, a gene encoding a protein to be expressed, and (iii)a 3′ untranslated terminator region.

A. Chimeric Promoter

Chimeric promoters are described above. In one embodiment, where thegene is employed in protein production in a monocot cell culture,preferred promoters are chimeric RAmy3E and RAmy3D gene promotersdescribed above. In another embodiment, where the gene is employed inprotein production in germinating seeds, a preferred promoter is achimeric RAmy1A gene promoter containing one or more sugar-depletionresponsive elements described above, to permit regulation control byboth gibberellic acid and sugar concentration during seed germination.

B. Expressed Protein Gene

The gene encoding the protein to be expressed may include a) the monocotα-amylase gene which is under the control of the native promoter fromwhich the improved promoter is derived; b) a gene encoding a monocotα-amylase normally controlled by a different promoter, or a geneencoding a monocot protein other than α-amylase; or c) a gene encoding aprotein from a source other than a monocot plant. The proteins ofcategories b) and c) are collectively referred to as heterologousproteins.

The expressed protein, if other than a monocot α-amylase, may include afusion of an N-terminal region corresponding to a portion of a monocotα-amylase signal sequence peptide and, immediately adjacent to theC-terminal amino acid of said portion, the protein. Suitable monocotα-amylase signal sequence peptides include, but are not limited to,those described in PCT publication WO 95/14099, incorporated byreference herein.

The expressed protein, with or without adjacent signal sequence, mayinclude therapeutic proteins such as factor VIII or α1-antitrypsin(AAT); vaccines; industrial enzymes such as subtilisin; and proteins andpeptides of nutritional importance. The nucleic acid coding sequence forthese proteins are available from a variety of reference and sequencedatabase sources. The coding sequence in a fusion protein gene isconstructed such that the final codon in the signal sequence isimmediately followed by the codon for the N-terminal amino acid of themature form of the protein to be expressed. The coding sequence for aheterologous protein may be codon-optimized for optimal expression inplant cells.

C. 3′ Untranslated Region

The gene also includes, downstream of the coding sequence, the 3′untranslated region (3′ UTR) from an inducible monocot gene, such as oneof the monocot α-amylase genes listed above. The transcriptionaltermination region may be selected, particularly for stability of themRNA to enhance expression. Polyadenylation tails, Alber and Kawasaki,Mol. and Appl. Genet. 1:419-434 (1982) are also commonly added to theexpression cassette to optimize high levels of transcription and propertranscription termination, respectively. Polyadenylation sequencesinclude but are not limited to the Agrobacterium octopine synthetasesignal, Gielen, et al., EMBO J. 3:835-846 (1984) or the nopalinesynthase of the same species Depicker, et al., Mol. Appl. Genet.1:561-573 (1982).

IV. Plant Transformation

For transformation of plants, the chimeric gene comprising the chimericpromoter of the present invention, a gene encoding a protein to beexpressed, and a 3′ untranslated terminator region, is placed in asuitable expression vector designed for operation in plants. The vectorincludes suitable elements of plasmid or viral origin that providenecessary characteristics to the vector to permit the vectors to moveDNA from bacteria to the desired plant host. Suitable transformationvectors are described in related application PCT WO 95/14099, publishedMay 25, 1995, which is incorporated by reference herein.

A. Transformation Vector

Vectors containing the improved promoter of the present invention mayalso include selectable markers for use in plant cells, such as thenptII or hph genes, for selection in kanamycin- orhygromycin-containingmedia, respectively, or thephosphinothricinacetyltransferasegene, for selection in mediumcontaining phosphinothricin (PPT).

The vectors may also include sequences that allow their selection andpropagation in a secondary host, such as sequences containing an originof replication and a selectable marker such as antibiotic or herbicideresistance genes, e.g., HPH (Hagio et al., Plant Cell Reports 14:329(1995) and van der Elzer, Plant Mol Biol. 5:299-302 (1985). Typicalsecondary hosts include bacteria and yeast. In one embodiment, thesecondary host is Escherichia coli, the origin of replication is acolE1-type, and the selectable marker is a gene encoding ampicillinresistance. Such sequences are well known in the art and arecommercially available as well (e.g., Clontech, Palo Alto, Calif.;Stratagene, La Jolla, Calif.).

The vectors which contain the chimeric promoter of the present inventionmay also be modified to intermediate plant transformation plasmids thatcontain a region of homology to an Agrobacterium tumefaciens vector, aT-DNA border region from Agrobacterium tumefaciens, and chimeric genesor expression cassettes (described above). Further, the vectors of theinvention may comprise a disarmed plant tumor inducing plasmid ofAgrobacterium tumefaciens.

B. Transformation of Plant Cells

The plants used in the process of the present invention are derived frommonocots, particularly the members of the taxonomic family known as theGramineae. This family includes all members of the grass family of whichthe edible varieties are known as cereals. The cereals include a widevariety of species such as wheat (Triticum sps.), rice (Oryza sps.)barley (Hordeum sps.) oats, (Avena sps.) rye (Secale sps.), corn (Zeasps.) and millet (Pennisettum sps.). In the present invention, preferredfamily members are rice and barley.

Plant cells or tissues derived from the members of the family aretransformed with expression constructs (i.e., plasmid DNA into which thechimeric gene of the invention has been inserted) using a variety ofstandard techniques (e.g., electroporation, protoplast fusion,Agrobacterium infection, or microparticle bombardment). In the presentinvention, particle bombardment is the preferred transformationprocedure.

Various methods for direct or vectored transformation of plant cells,e.g., plant protoplast cells, have been described, e.g., in above-citedPCT application WO 95/14099. As noted in that reference, promotersdirecting expression of selectable markers used for plant transformation(e.g., nptII) should operate effectively in plant hosts. One suchpromoter is the nos promoter from native Ti plasmids, Herrera-Estrella,et al., Nature 303:209-213 (1983). Others include the 35S and 19Spromoters of cauliflower mosaic virus, Odell, et al, Nature 313:810-812(1985), and the 2′ promoter, Velten, et al., EMBO J. 3:2723-2730 (1984).

In one preferred embodiment, the embryo and endosperm of mature seedsare removed to exposed scutellum tissue cells. After propagatingscutellar tissue into callus, the callus cells may be transformed by DNAbombardment or injection, or by vectored transformation, e.g., byAgrobacterium infection after bombarding the scutellar cells withmicroparticles to make them susceptible to Agrobacterium infection(Bidney et al., Plant Mol Biol. 18:301-313 (1992)).

One preferred transformation follows the methods detailed generally inSivamani, E. et al., Plant Cell Reports 15:465 (1996); Zhang, S., etal., Plant Cell Reports 15:465 (1996); and Li, L., et al., Plant CellReports 12:250 (1993).

V. Cell Culture Production of Expressed Protein

In another aspect, the invention relates to expression of recombinantproteins in cereal cell culture, the expression regulated by thechimeric promoter of the present invention. In one embodiment of theinvention, the cells are derived from scutellar epithelium of cerealplants. Example 1A outlines a preferred method for initiation ofscutellar callus and suspension cultures. The chimeric promoters,vectors and methods described above may be implemented in the practiceof this aspect of the invention.

Transgenic plant cells which express the recombinant protein under thecontrol of the chimeric promoter of the present invention are culturedunder conditions that favor plant cell growth, until the cells reach adesired cell density, then under conditions that favor expression of theprotein under the control of the chimeric promoter.

For example, rice cells transformed with an expression vector of thetype described above containing the sequence encoding humanα1-antitrypsin (AAT) fused to the coding sequence for the Amy3D signalsequence, under the control of a chimeric Amy3D promoter, are grown inshake flasks (or in a bioreactor) using AA2 growth media containing 3%sucrose (AA2+sucrose). Cells are cultured at 28° C. in the dark withconstant shaking for aeration. The medium is replenished every five daysto maintain healthy cell growth. The pH of the media is maintained at pH5.7. Since the AAT coding sequence is under the control of asugar-depletion responsive promoter (chimeric Amy3D), recombinant AATproduction is induced by diluting or changing the media to sucrose-freemedia (AA2-sucrose). Upon induction, recombinant AAT is expressed in thecells and secreted into the culture medium by virtue of the Amy3D signalpeptide. Recombinant AAT is isolated from the culture medium andpurified to homogeneity using standard techniques known to those ofskill in the art.

VI. Production of Expressed Protein in Germinating Seeds

In this embodiment, monocot cells transformed as above are used toregenerate plants, seeds from the plants are harvested and thengerminated, and the mature protein is isolated from the germinatedseeds.

Plant regeneration from cultured protoplasts or callus tissue is carriedby standard methods, e.g., as described in Evans et al., HANDBOOK OFPLANT CELL CULTURES Vol. 1: (MacMillan Publishing Co. New York, 1983);and Vasil I. R. (ed.), CELL CULTURE AND SOMATIC CELL GENETICS OF PLANTS,Acad. Press, Orlando, Vol. 1, 1984, and Vol. 111, 1986, and as describedin the above-cited PCT application.

The transgenic seeds obtained from the regenerated plants are harvested,and prepared for germination by an initial stepping step, followed bymalting as detailed, for example, in above-identified PCT application WO95/14099.

The mature protein secreted from aleurone cells into the endospermtissue of the seed can be isolated by standard methods. Typically, theseeds are mashed to disrupt tissues, the seed mash is suspended in aprotein extraction buffer, and the protein is isolated from the bufferby conventional means.

Stably transformed transgenic cereal seeds prepared as described abovemay also be used as a source of homogenous populations of callus cells,which can be cultured to produce the recombinant protein in cellculture.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters which could be changed or modified to yieldessentially similar results.

EXAMPLES

General Methods

Generally, the nomenclature and laboratory procedures with respect tostandard recombinant DNA technology can be found in Sambrook, et al.,Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. 1989 and in S. B. Gelvin and R. A. Schilperoot,Plant Molecular Biology, 1988. Other general references are providedthroughout this document. The procedures therein are known in the artand are provided for the convenience of the reader.

Example 1 Expression of β-glucuronidase Under the Control of theα-amylase Promoter in Rice Cell Culture

A. Initiation of Scutellar Callus and Suspension Cultures.

Rice seeds (Oryza sativa L. cv. M202) were provided by Dr. M. Brandon(California Rice Experimental Station). Seeds were dehulled, washedthree times with water, rinsed in 70% ethanol for 20 sec and thensurface-sterilized in 1% sodium hypochlorite with a few drops of Tween20 under vacuum for 20 min. Sterilized seeds were washed three timeswith sterile distilled water. Seven seeds were placed in 15 cm petridishes containing LS medium with 2 mg/l 2,4-D and 30 g/l sucrose. Theseeds were incubated in the dark at 28° C. and checked periodically tomonitor the growth of scutellar-derived callus. Callus formation fromscutellum tissue and/or embryo was visible after 5 days. After 30 to 40days, clumps of friable calli, about 1 cm in diameter, were saved andthe remaining tissue was discarded.

To initiate a suspension culture, friable calli were gently agitated ina petri dish with liquid AA medium as described by Thompson J A,Abdullah R and Cocking E C, Protoplast culture of rice (Oryza sativa L.)using media solidified with agarose (Plant Sci. (1986) 47:123-133) toreduce the calli to small clusters of cells. Cell clusters from about20-30 clumps of calli were then transferred to a 125 ml Erlenmeyer flaskand the liquid was replaced with 25 ml of fresh AA medium. The flaskswere incubated in the dark on a rotary platform shaker at 110 rpm and28° C. The primary culture was sub-cultured every 4 to 5 days withrepeated screening for small cell clusters. This was accomplished bypassing the culture sequentially through nylon filters of 1000 μm and500 μm pore size. After two months of subculture, a finely divided andrapidly growing suspension culture was obtained. This culture wassubsequently maintained by weekly subculture in AA medium containing 3%sucrose.

B. Construction of RAmy3D/GUS Gene Fusion Plasmids

The RAmy3D promoter/GUS gene fusion expression plasmid shown in FIG. 2Awas constructed in three steps. First, a 1.5 kb SalI fragment containingthe promoter and part of the coding region from rice genomic cloneλOSg1A as described by Huang, et al., (1990; Nucleic Acid Res.,18:7007-7014) was subcloned into pBluescript KS-to produce the plasmidp1AS1.5. The Alul fragment from p1AS1.5 containing 876 bp of promoterand 66 bp of 5′ untranslated region was subcloned into the EcoRV site of“pBLUESCRIPT KS+” to form p1Alu.

Second, a plasmid containing a promoterless GUS cassette was constructedby subcloning the HindIII/EcoRI GUS cassette from pB11O1 (Jefferson R A,(1987a) Plant Mol. Biol. Reporter, 5:387-405) into pUC19 to form pB1201.A pUC19 polylinker in front of the GUS coding region provides convenientcloning sites for inserting promoter fragments. Third, the RAmy3Dpromoter fragment was inserted into the promoterless GUS plasmid toproduce the plasmid p3DG. The Xbal/Alul (in HindIII site) promoterfragment from p1Alu was ligated into Xbal/Smal digested pB1201. Thefinal 11 bp of RAmy3D 5′ untranslated region was substituted by 21 bpfrom the polylinker resulting in the 5.83 kb plasmid, p3DG.

The junction between the RAmy3D promoter and the 5′ end of the GUS genewas confirmed by DNA sequencing. DNA restriction digest, DNA gelelectrophoresis, ligation, transformation, plasmid DNA isolation and DNAsequencing followed standard procedures (Sambrook, et al., (1989)supra).

To prepare 5′ deletions of the RAmy3D promoter, plasmid pDG3 wasdigested with PstI and XbaI for constructing a 5′ unidirectionaldeletion series using the Bluescript Exo/Ming DNA Sequencing System(Stratagene). The deletion endpoints were located by DNA sequencingusing the dideoxy method, The predicted transcription start site of theRAmy3D gene was used as the reference point for identifying locationswithin the promoter sequence.

Mutations were introduced into specific regions of the RAmy3D promoterin p3DG using standard oligonucleotide-directed in vitro mutagenesismethods. To facilitate screening of putative mutants, restrictionendonuclease sites were included in the mutagenic sequences. Allsite-directed mutations (SDM) were confirmed by sequencing using thedideoxy method (Amersham Life Sciences).

Plasmid p3DGB, which was prepared by inserting a bar gene cassette intop3DG, was the vector used in the transient expression experiments. ANotI fragment containing 35S promoter/bar coding region/Nos 3′ regionwas cut from pBARGUS (Fromm, M. E. (1990) Bio/Technology 8:833-839),blunted with T4 DNA polymerase and inserted into the Smal site ofpBluescript SK(−) to make pBSB. The bar gene cassette was then cut outof pBSB by double digestion with XbaI and HindIII and inserted intosimilarly digested pUC19 to form pUCB. The bar gene cassette was thencut from pUBC as an EcoRI fragment and ligated into the EcoRI site ofp3DG and each of the site-directed Amy3D mutant constructs for transientexpression analysis.

C. Preparation of Transgenic Rice Cell Lines and Analysis of Amy3D/GUSGene Expression

Plasmids were transformed into rice cells by microprojectile bombardmentusing the Biolistic PDS-1000/He (BioRad Laboratories) with 9 MPa (1300psi) rupture disks. Suspension culture cells for bombardment werelayered 1-2 mm thick on sterile filter paper disks on AA2 agarmedium(ref 52) and were incubated in the dark at 28° C. for 5 days.

Target cells were cotransformed by gold particles of 1 μm in diametercoated with 2.5 μg of pMON410 (containing the hph gene driven by theCaMV 35S promoter; Rogers S. G. et al. (1987) Meth. Enzymol.153:253-277) and 2.5 μg of the desired promoter/GUS plasmid (for 6bombardments). Plates to be bombarded were placed 8 cm below thestopping plate under vacuum at 95 kPa (28 in. Hg). The bombarded cellswere incubated in the dark at 28° C. for 5 days without selectionpressure and transferred to AA2 agar medium containing 30 mg/lhygromycin B. After 2 weeks, cells were transferred to medium containing50 mg/l hygromycin B. Transfer of cells to fresh AA2 medium containing50 mg/l hygromycin B was done every 2 weeks until hygromycin-resistantcalli reached 1 cm in diameter.

Transgenic callus clones containing the promoter/GUS fusion constructswere identified by GUS histochemical assay (Jefferson, R. A. et al.(1987b) EMBO J. 6:3901-3907) or by PCR amplification of template DNAfrom the transgenic callus using forward and reverse primers whichanneal to the 5′ untranslated region of the Amy3D gene and to the GUScoding region, respectively.

Transgenic suspension cells were washed in two changes of basal mediumplus 330 mM mannitol. Washed cells were then incubated in basal mediumplus various chemicals for 2 days at 110 rpm on a rotary shaker at 28°C. GUS enzyme was extracted from the cells and assayed by thefluoromteric method (Jefferson et al. (1987b), supra) except that thecells were disrupted by sonication for 3 min at 50% duty cycle and 10microtip limit on the Branson Sonifier 450. Transgenic cell linescontaining the promoterless construct (RAmy3D promoter deleted to +40)were used as a negative control to define background levels of GUSactivity.

D. Protoplast Isolation and Transient Expression Assays

Protoplast methods were modified from Lee et al. (Planta 178:325-333(1989)). After removing AA2 medium, 7-day old suspension cells of ricecv. M202 were incubated with 40 ml of cell wall degrading enzymesolution (0.5% Onozuka Cellulase RS, 0.5% Macerozyme R-10; Yakult HonshaCo.), 5 mM MES, 0.32 M mannitol, 0.08 M glucose with CPW salts)overnight with 50 rpm shaking at 28° C. in the dark.

Digested cells were filtered through a 149 μm nylon filter and thenthrough a 20 μm nylon filter, the filtrate was centrifuged for 12 min at800 rpm, and gently resuspended in the same volume of CPW salts solutionwith 0.4M mannitol, pH 5.8.

Protplasts were centrifuged at 800 rmp for 7 min and resuspended in ASPbuffer (70 mM aspartic acid monopotassium salt, 5 mM calcium gluconate,5 mM MES and 0.4 M mannitol, pH 5.8) at a concentration of 10 millionprotoplasts/ml.

A 0.8 ml volume of protoplasts were mixed with 50 μg of p3DGB (orsite-directed mutants of RAmy3D in p3DGB), 50 μg of pMef plasmid DNA(carrying the CaMV 35S promoter/luciferase gene fusion; Li et al. (1993)Plant Cell Rep. 12:250-255) and incubated on ice. After 30 min,protoplasts were transferred to an electroporation cuvette (0.4 cmelectrode gap) and electroporated with a “GENE PULSER” electroporator(Bio-Rad) at 600 volt/cm, 500 μF, time constant 50 ms.

After incubation on ice for an additional 30 min, the protoplasts weretransferred to room temperature for 30 min. The ASP buffer was removedby centrifugation. Protoplasts were resuspended in Kao and MichaylukMedium (Kao KN (1977) Mol. Gen. Genet. 150:225-230; Sigma ChemicalCorp.) modified by removal of sugars, casamino acids and coconut water.For sugar-depletion experiments, 0.4 M mannitol was added to thismedium, and for glucose treatments, 0.08 M glucose plus 0.32 M mannitolwas added. Protoplasts were incubated for 2 days at 28° C. in the darkwithout shaking. After incubation, the protoplasts were harvested bycentrifuging at 800 rpm for 5 min. After removing the supernatant, 100μl of GUS/LUX extraction buffer (0.1 M potassium phosphate monobasic, 1mM EDTA and 7 mM 2-mercaptoethanol) was added and cells were sonicatedfor 3 min at 10% duty cycle and 10 microtip limit settings on theBranson Sonifier 450. Sonicated protoplasts were centrifuged at 14,000rpm for 5 min. The supernatant was used to measure the GUS and LUXactivities (Bronstein, I. et al.(1994) Biotechniques 17:172-178; deWetJ. R. et al.(1987) Mol. Cell Biol. 7:725-737).

E. Identification of Sugar-Depletion Responsive Elements

E1. Deletion Analysis

GUS expression in transformed rice cell lines was examined using plasmidconstructs having the series of nested deletions in the Amy3D promotershown in FIG. 3A. The uppermost three promoter deletions shown in thefigure (−754, −558, and −346) all contain the identified promoterconsensus regions: pyrimidine element; three CGSCG elements; the GC-richelement; the G box; the Amy element; and the TATA box. These threedeletion constructs gave levels of GUS expression under sugar-depletionconditions (e.g., in the presence of 0.4 M mannitol) essentiallyequivalent to that level of expression seen under the same conditionswith the full-length promoter (−876; FIG. 3B).

In the series of deletions proceeding from the 5′ end of the Amy3Dpromoter, as diagrammed in FIG. 3A, the first sharp decline in sugardepletion-induced GUS expression occurred upon deletion of the regionbetween nucleotides −206 and −98 (FIG. 3B), indicating that this regioncontains sequence critical for the sugar-depletion response of thepromoter.

E2. Mutation Analysis

The −207 to −98 region was examined in greater detail using full-lengthAmy3D promoters containing short mutated segments SDM1-SDM10 as shown inFIG. 4A. Levels of GUS expression, as a function of mutated promotersegment, were measured in the transient expression assay system and theresults shown in FIG. 4B. Levels of sugar-depletion induction comparableto that of the wild-type 3RAmy3D promoter were seen for all of themutated segments except those labeled SDM5, SDM6, and SDM8. These threesegments reside between nucleotides −166 and −125 of the Amy3D promoter,which has the sequence CGACG CGGCG CCTAC GTGGC CATGC TTTAT TGCCT TATCCAT identified herein as SEQ ID NO:1. The three segments are identifiedas: a) SDM5, having the sequence CGACGCGGCG extending between −166 and−157, identified as SEQ ID NO:2; b) SDM6, having the sequence CTACGTGGCextending between −155 and −147, and identified herein as SEQ ID NO:3;and c) SDM8, having the sequence TTATCCAT extending between −132 and−125 and identified herein as SEQ ID NO:4.

The region −165 to −146, which contains the SDM5 and SDM6 sequenceelements, was subjected to more detailed mutational analysis as shown inFIG. 5A. Within this region, a series of five SDM5 and SDM6site-directed mutants were tested for GUS expression in the transientexpression system, with the results shown in FIG. 5B. Significantreduction in GUS expression was observed in mutations of RAmy3D in thesegment corresponding to SDM5-1 and in all of three of the SDM6segments.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

1. A chimeric plant promoter for upregulating expression of a codingsequence operatively linked to the promoter, under conditions of sugardepletion or deprivation in a plant cell, comprising: (i) a promoterelement effective to express the coding sequence in plant cells, underselected conditions, and (ii) carried in said promoter element, aheterologous sequence from the Amy3D promoter comprising SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or combinationsthereof.
 2. The chimeric promoter of claim 1, wherein the heterologoussequence is identified by SEQ ID NO:
 1. 3. The chimeric promoter ofclaim 1, wherein the heterologous sequence is identified by SEQ ID NO:2.
 4. The chimeric promoter of claim 1, wherein the heterologoussequence is identified by SEQ ID NO:
 3. 5. The chimeric promoter ofclaim 1, wherein the heterologous sequence is identified by SEQ ID NO:4.
 6. The chimeric promoter of claim 1, wherein the heterologoussequence is SEQ ID NO:
 5. 7. The chimeric promoter of claim 1, whereinthe heterologous sequence is duplicated.
 8. The chimeric promoter ofclaim 1, wherein the promoter element is from a monocot α-amylase geneselected from the group consisting of the RAmy1A, RAmy1B, RAmy2A,RAmy3A, RAmy3B, RAmy3C, pM/C, gKAmy141, gKAmy155, Amy32b, and HV18genes.
 9. A method of enhancing the inducibility by sugar depletion ordeprivation of a plant or plant-virus promoter in plant cells, saidmethod comprising: introducing into a plant promoter or plant-viruspromoter, a heterologous sequence from the Amy3D promoter comprising SEQID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5 orcombinations thereof, by said introducing, achieving at least a 2-foldgreater sugar-depletion induction level of the promoter in plant cellsover that of the unmodified promoter.
 10. The method of claim 9, whereinthe promoter element is from an α-amylase gene selected from the groupconsisting of the RAmy1A, RAmy1B, RAmy2A, RAmy3 A, RAmy3 B, RAmy3C,pM/C, gKAmy141,gKAmy155, Amy32b, and HV18 genes.
 11. The method of claim9, wherein the heterologous sequence is duplicated in the chimericpromoter.
 12. The method of claim 9, wherein the plant promoter is froma monocot plant.
 13. A vector for use in tranforming a plant,comprising: a chimeric gene having, operatively linked in sequence in a5′ to 3′ direction, (i) the chimeric plant promoter of claim 1, (ii) agene encoding a protein to be expressed, and (iii) a 3′ untranslatedterminator region.
 14. Plant cells transformed with the vector of claim13.
 15. The plant cells of claim 14, which are monocot cells.